Kojibiose (2-O-α-D-glucopyranosyl-D-glucopyranose) is a promising prebiotic for the stimulation of beneficial bacterial populations in the human gut1, 2. The α-1,2 bond can indeed be handled by specific micro-organisms such as Bifidobacteria, but it is largely resistant to the action of enzymes in the digestive tract3, 4. Moreover, kojibiose and derivatives have the ability to inhibit α-glucosidase I in different tissues and organisms5-8. Accordingly, these are appealing molecules because inhibitors that act upon α-glucosidase I are suggested to be interesting candidate drugs for the treatment of human immunodeficiency virus type 1 (HIV-1) infections9. Given their α-glucosidases inhibitory action, they can also limit the digestion of dietary carbohydrates and could therefore be useful to counter diabetes, obesity and cardiovascular diseases10, 11. Studies on the health-promoting properties of kojibiose are however hampered by the limited availability of this disaccharide1. Kojibiose is naturally present in honey12, beer13, sweet potato starch hydrolysate14, sake and koji15 (hence the name), but the amounts are too low for extraction on larger scale. According to some authors, the isolation from the partial acetolysis of dextran from Leuconostoc mesenteroides NRRL B-1299 is currently the best method4, 16, 17. Yet, it is a multi-step process involving several chemical reagents like glacial acetic acid, acetic anhydride, concentrated sulphuric acid, chloroform and methanol. Chemical synthesis via modified Koenigs-Knorr reactions is still applied as well, despite the need for multiple (de)protection steps, silver or mercury catalysts and solvents like acetonitrile, and low yields18-20. Alternatively, enzymes like α-glucosidase21, 22, α-glucoamylase22, 23, glucansucrase24, dextransucrase25 and kojibiose phosphorylase4 can be used, but unfortunately these often have low yields and/or generate product mixtures. Sucrose phosphorylase (SP) is another enzyme that is capable of producing kojibiose26, 27, starting from sucrose (renewable, cheap and readily available28) and D-glucose. It naturally catalyses the reversible phosphorolysis of sucrose into α-D-glucose 1-phosphate and D-fructose, but it can also glycosylate alternative acceptors with yields exceeding 90%29, 30 (WO 2011/124538). In case of D-glucose as acceptor, the overall kojibiose yield is however lowered by the side-formation of maltose (4-O-α-D-glucopyranosyl-D-glucopyranose) as glucobiose product27. There is thus a need to produce a, preferably thermostable, sucrose phosphorylase having a high selectivity of kojibiose production.